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American Journal of Clinical Nutrition, Vol. 86, No. 2, 360-365, August 2007
© 2007 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Lysine requirement of healthy school-age children determined by the indicator amino acid oxidation method1,2,3

Rajavel Elango, Mohammad A Humayun, Ronald O Ball and Paul B Pencharz

1 From the Research Institute, The Hospital for Sick Children, Toronto, Canada (RE, MAH, and PBP); the Department of Nutritional Sciences, University of Toronto, Canada (PBP and ROB); and the Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada (ROB and PBP)

2 Supported by the Canadian Institutes for Health Research (grant MT 10321). Mead Johnson Nutritionals (Canada) donated the protein-free powder for the experimental diets.

3 Address reprint requests and correspondence to PB Pencharz, Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail: paul.pencharz{at}sickkids.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The current Dietary Reference Intake (DRI) recommendations for lysine requirements in children are based on a factorial estimate.

Objective: The objective of the current study was to determine the lysine requirement in healthy school-age children by measuring the oxidation of L-[1-13C]phenylalanine to 13CO2 (F13CO2) in response to graded intakes of lysine.

Design: Five healthy school-age children randomly received each of 7 lysine intakes (5, 15, 25, 35 50, 65, and 80 mg · kg–1 · d–1) along with an amino acid mixture to give a final calculated protein intake of 1.5 g · kg–1 · d–1 and an energy intake of 1.7 x resting energy expenditure (REE). The mean lysine requirement was determined by applying 2-phase linear regression crossover analysis on F13CO2 data, which identified a breakpoint (requirement) at minimal F13CO2 in response to graded lysine intakes.

Results: The mean and population-safe (upper 95% CI) lysine requirements were determined to be 35 and 58 mg · kg–1 · d–1, respectively.

Conclusions: The mean and population-safe lysine requirements for children are similar to those for adults (36 and 52 mg · kg–1 · d–1, respectively), which suggests that the findings from the current study reflect predominantly the maintenance lysine requirements in children and not all requirements for growth. Therefore, to ensure age-appropriate growth in school-age children, we propose the addition of the requirement of lysine for growth ({approx}6 mg · kg–1 · d–1) to the mean estimate. The new mean and population-safe lysine requirements are 41 and 58 mg · kg–1 · d–1, respectively; these values are significantly higher than the current DRIs of 37 and 46 mg · kg–1 · d–1, respectively.

Key Words: Lysine • indicator amino acid oxidation • amino acid requirements • stable isotopes • children


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysine is an indispensable amino acid that is required preformed in the diet of humans (1). Lysine is also the first limiting amino acid for protein synthesis in persons consuming a predominantly cereal-based diet such as wheat and rice (1). The current requirement for lysine in children proposed by the Institute of Medicine, Food and Nutrition Board, in the recent Dietary Reference Intakes (DRIs) for macronutrients (2), is based on a factorial approach because of the lack of conclusive empirical data. The only previous study of lysine requirements in children aged 10–12 y is one by Nakagawa et al in 1961 (3), who used the nitrogen balance technique. They reported a mean lysine requirement of 60 mg · kg–1 · d–1 in children aged 10 y. This estimated lysine requirement was considered an overestimate by the Institute of Medicine because the children had growth rates {approx}5-fold higher, at adequate lysine intakes, than those predicted for children in this age group.

No studies of lysine requirements in children with the use of newer stable-isotope methods have been reported. To accurately determine amino acid requirements, it is necessary to provide subjects with a range of amino acid intakes including zero or very low test amino acids. Because of ethical reasons, children cannot be fed these very low test amino acid diets for prolonged periods of time. The minimally invasive indicator amino acid oxidation (IAAO) method, initially developed in adults (4) in our laboratory to determine amino acid requirements, overcomes this problem. The IAAO method takes 8 h to conduct: adaptation to the test diet for 6 h and then breath sampling for 2 h. This method has been applied to determine all of the adult indispensable amino acid requirements (5-11) and was reviewed in detail earlier by Zello et al (12) and recently by Pencharz and Ball (13). In children, the initial application of this method was in patients with phenylketonuria to determine their tyrosine (14) and phenylalanine (15) requirements. Recently, we also applied the IAAO method to healthy school-age children to determine total branched-chain amino acid (BCAA) (16), total sulfur amino acid (TSAA) (17), and minimum methionine (18) requirements.

The objective of the current study was to determine the lysine requirements in healthy school-age children with the IAAO method. We also wanted to compare the results from the current study with those of our previous estimates of lysine requirements in adults, because our previously derived estimated BCAA and TSAA requirements in children were similar to those for adults.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Five healthy school-age children (4 boys and 1 girl) participated in the study at the Clinical Investigation Unit, The Hospital for Sick Children (SickKids), Toronto, Canada. Subject characteristics, body composition, and energy intakes are summarized in Table 1Go. The children participating in the study had no recent history of weight loss or illness and none used any medication during the study period. Written informed consent was obtained from the parent or guardian, and the assent of the participating children was also obtained. The parent or guardian of each participating child received financial compensation for the costs incurred while participating in the studies. All procedures in the study were approved by the Research Ethics Board at SickKids.


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  		TABLE 1 Characteristics and energy intake of children participating in the study1

 
Experimental design
The experimental design was based on the minimally invasive IAAO model developed in adults (4) and recently used in healthy children (16-18). Before the studies began, all children fasted overnight ({approx}12 h) and were brought to the Clinical Investigation Unit for body-composition analysis and resting energy expenditure (REE) measurements. Body composition was measured by bioelectrical impedance analysis (model 101A; RJL Systems, Detroit, MI). REE was measured by continuous, open-circuit indirect calorimetry (Deltatrac II Metabolic Monitor; SensorMedics, Yorba Linda, CA). Two days before the study day, the children were prescribed a maintenance diet. The maintenance diet was prescribed to each subject on the basis of 3-d food records and provided energy at 1.7 x REE and protein at 1.5 g · kg–1 · d–1. The menus consisted of typical foods consumed by the children, and food records were collected to ensure the consistency of dietary intakes. On the study day, after a 12-h fast, the subjects were randomly assigned to receive 1 of 7 test lysine intakes: 5, 15, 25, 35, 50, 65, or 80 mg · kg–1 · d–1. The alanine intake was adjusted with varying lysine intakes to maintain a constant nitrogen intake. The study day diet was consumed as 8 hourly isonitrogenous and isocaloric meals; each meal represented one-twelfth of the daily requirement (Figure 1Go). During the study day, the subjects were not allowed to consume anything else except water.


Figure 1
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  		FIGURE 1. Study-day protocol for each indicator amino acid. The experimental diet was given as a test meal hourly for 8 h. Each meal was isocaloric and isonitrogenous and represented one-twelfth of each subject's daily requirement. Priming doses of the isotopes NaH13CO3 and L-[1-13C]phenylalanine were given at the fifth meal; an hourly dose of L-[1-13C]phenylalanine was given simultaneously and continued throughout the remaining 4 h. Three baseline breath samples were collected 45, 30, and 15 min before the tracer protocol began, and 2 baseline urine samples were collected 45 and 15 min before the tracer protocol began. Four plateau breath and urine samples were collected at isotopic steady state every 30 min, beginning 2.5 h after the tracer protocol began. The carbon dioxide production rate (Formula 3CO2) was measured by indirect calorimetry after the fifth hourly meal.

 
Tracer protocol
On each study day the subjects consumed 4 hourly meals before the oral tracer infusion protocol (Figure 1Go). A priming dose of 2.07 µmol/kg NaH13CO3 (99 atom% excess; Cambridge Isotope Laboratories, Woburn, MA) was given at the fifth meal. A prime (6.55 µmol/kg) and hourly (11.8 µmol · kg–1 · h–1) dose of L-[1-13C]phenylalanine (99 atom% excess; Cambridge Isotope Laboratories, Woburn, MA) was simultaneously given at the fifth meal and on an hourly basis with subsequent meals until the end of the study. The quantity of phenylalanine supplied as L-[1-13C]phenylalanine during the last 4 h of the study was subtracted from the diet to provide a total intake of 25 mg phenylalanine · kg–1 · d–1. Tyrosine was provided at 61 mg · kg–1 · d–1 to ensure an excess of tyrosine. All subjects participated in all 7 studies, and each study was separated by ≥1 wk.

Study diets
The experimental diet consisted of a protein-free powder (Product 80056; Mead Johnson, Evansville, IN) flavored with Tang and Kool-Aid (Kraft Foods, Toronto, Canada), corn oil, a crystalline L-amino acid mixture (based on egg protein composition to provide protein at 1.5 g · kg–1 · d–1), and protein-free cookies. Energy was provided at 1.7 x REE on the basis of each subject's measured REE after a 12-h fast, as described earlier. The diet provided 53% of the energy as carbohydrate, 37% as fat, and 10% as protein. The subjects also consumed a daily multivitamin supplement (Centrum Junior; Whitehall-Robins, Mississauga, Canada) for the duration of all 7 studies.

Sample collection and analysis
Breath and urine samples were collected on all study days. Previously, Bross et al (4), during the development of the minimally invasive IAAO model, showed that urinary and plasma enrichments of L-[1-13C]phenylalanine are similar at isotopic steady state. Three baseline breath samples were collected at 45, 30, and 15 min, and 2 baseline urine samples were collected 45 and 15 min before the tracer protocol began (Figure 1Go). Four plateau breath and urine samples were collected at isotopic steady state every 30 min, beginning 2.5 h after the start of the tracer protocol. The CV between the 4 plateau values of enrichment was <5%. Breath samples were collected in disposable Haldane-Priestly tubes (Venoject; Terumo Medical, Elkton, MD) by using a collection mechanism that permits the removal of dead-space air. Breath samples were stored at room temperature and urine samples at –20 °C until analyzed. During each study day, the rate of carbon dioxide production was measured immediately after the fifth meal for 20 min with an indirect calorimeter (Deltatrac II Metabolic Monitor; Sensormedics, Yorba Linda, CA).

Expired 13CO2 enrichment was measured by continuous-flow isotope ratio mass spectrometry (CF-IRMS20/20 isotope analyzer; PDZ Europa Ltd, Cheshire, United Kingdom). Enrichments were expressed as atoms percent excess (APE) compared with a reference standard of compressed carbon dioxide gas. Urinary L-[1-13C]phenylalanine enrichment was analyzed with a triple quadrupole mass spectrometer (API 4000;Applied Biosystems-MDS SCIEX, Concord, Canada) coupled to an Agilent 1100 HPLC system (Agilent Technologies Canada Inc, Mississauga, Canada) as described previously (17). Briefly, a 62.5-µL urine aliquot was deproteinized with 200 µL methanol and centrifuged at 7000 x g for 5 min. The supernatant fluid was freeze-dried and reconstituted in 1 mL 0.1% formic acid. The amino acids were separated with a Waters Xterra MS C18 3.5 µm 2.1 x 150 mm column (Waters Corp, Milford, MA) with a binary LC gradient (40–60% aqueous acetonitrile containing 0.1% formic acid). L-[1-13C]Phenylalanine enrichment was then analyzed with the triple quadrupole mass spectrometer (API 4000) operated in positive ionization mode. Selected ion chromatograms were obtained by monitoring the mass-to-charge ratios of the product ions of 165 and 166 for [1-13C]phenylalanine corresponding to the unenriched (M) and enriched (M + 1) peaks, respectively. The areas under the peaks were integrated by ANALYST NT software (version 1.4.1; Applied Biosystems-MDS SCIEX). Isotopic enrichment was expressed as molecules percent excess and calculated from peak area ratios at isotopic steady state at baseline and plateau.

Estimation of isotope kinetics
Whole-body phenylalanine flux was calculated from the dilution of L-[1-13C]phenylalanine in the body amino acid pool at isotopic steady state by using the following equation (9, 19):

Formula 1(1)
where Q is phenylalanine flux (in µmol · kg–1 · h–1), i is the rate of L-[1-13C]phenylalanine infused (µmol · kg–1 · h–1), and Ei and Eu are the isotopic enrichments as mole fractions (molecules percent excess) of the infusate and urinary phenylalanine, respectively, at isotopic plateau.

The rate of phenylalanine oxidation was calculated as follows:

Formula 2(2)
where O represents phenylalanine oxidation (µmol · kg–1 · h–1) and F13CO2 represents the rate of 13CO2 released by phenylalanine tracer oxidation (µmol · kg–1 · h–1) calculated by the following equation:

Formula 3(3)
where FCO2 is the carbon dioxide production rate (mL/min), ECO2 is the 13CO2 enrichment in expired breath at isotopic steady state (atoms percent excess), and W is the weight (kg) of the subject. The constants 44.6 (µmol/mL) and 60 (min/h) were used to convert FCO2 to µmol/h; 0.82 is the correction factor for carbon dioxide retained in the body due to bicarbonate fixation (20), and the factor 100 changes atom percent excess to a fraction.

Statistical analysis
Results are expressed as means ± SDs. A mixed linear model with subject as a random variable using PROC MIXED (SAS/STAT version 8.2, SAS Institute, Cary, NC) was used to analyze the effects of lysine intakes on F13CO2, phenylalanine flux, and oxidation. When warranted, a post hoc analysis was performed by using Tukey's multiple comparisons test. Differences were considered significant at P < 0.05.

Estimates of the mean lysine requirement for children were derived by breakpoint analysis of the F13CO2 data by using a 2-phase linear regression crossover model. This model selects for the minimum residual SE in a stepwise partitioning of data points between 2 regression lines. The first regression line has a slope, and the second line is horizontal with minimal or no slope. Breakpoint analysis was done by using the mixed and regression procedure in SAS program with subject as a random variable. The safe intake of lysine (upper 95% CI, equivalent to the Recommended Dietary Allowance) was calculated by using Fieller's Theorem (21). Briefly, 95%CI = breakpoint ± tdf,{alpha}/2 x SE, where SE is the SE of the breakpoint, df is the df associated with the residual mean square of the best fit model, and {alpha} is the 95% confidence level. The upper 95% CI was calculated by using SAS/STAT version 8.2 (SAS Institute).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subject characteristics
Five healthy school-aged children (8.4 ± 0.9 y) completed the study. Their anthropometric measures (Table 1Go) were within the normal range for age (22, 23) and did not change during the course of the study. Similarly, energy and protein intakes of the subjects were adequate. According to self- and parent-rated Tanner staging (24), all subjects were either in Tanner Stage I or II.

Phenylalanine flux and oxidation
Phenylalanine flux was not affected (P > 0.05) by different lysine intakes (Table 2Go) as required by the IAAO method. This result indicated that the precursor pool for the indicator amino acid oxidation did not change in size in response to the test amino acid (ie, lysine). Therefore, the changes in oxidation were inversely proportional to the changes in protein synthesis. Phenylalanine oxidation declined in response to graded increases in lysine intake, but only a lysine intake of 5 mg · kg–1 · d–1 differed significantly (P < 0.05) from the lysine intakes of 35, 65, and 80 mg · kg–1 · d–1 (Table 2Go).


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  		TABLE 2 Effect of lysine intake on phenylalanine flux and oxidation in healthy school-age children1

 
L[1-13C]Phenylalanine oxidation
The rate of 13CO2 released from the oxidation of L-[1-13C]phenylalanine (F13CO2) decreased in children with increasing lysine intakes up to 35 mg · kg–1 · d–1 (Figure 2Go). Further increases in lysine intake did not result in changes in individual F13CO2 values, which indicated no further increase in incorporation of label for protein synthesis. Two-phase linear regression crossover analysis of F13CO2 data resulted in the identification of a breakpoint for mean lysine requirement of 35 mg · kg–1 · d–1 (Figure 3Go). The mean lysine requirement represents the estimated average requirement (EAR). The upper 95% CI, which represents the population-safe or Recommended Dietary Allowance was determined to be 58 mg · kg–1 · d–1.


Figure 2
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  		FIGURE 2. Influence of dietary lysine intake on the production of 13CO2 from the oxidation of orally administered L-[1-13C]phenylalanine in healthy school-age children (n = 35 observations). Individual values for 4 boys and 1 girl at 7 lysine intakes are shown. The closed triangle represents the female subject.

 

Figure 3
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  		FIGURE 3. Influence of dietary lysine intake on the production of 13CO2 from the oxidation of orally administered L-[1-13C]phenylalanine in healthy school-age children (n = 35 observations). Values are means ± SDs at each test lysine intake. The breakpoint estimates the mean lysine requirement. The breakpoint was determined by using 2-phase linear regression crossover analysis to minimize the total sum of squares in error for the combined line. The mean lysine requirement was estimated to be 35 mg · kg–1 · d–1. The upper 95% CI estimates the population-safe intake and was estimated to be 58 mg · kg–1 · d–1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mean and population-safe lysine requirements for healthy school-age children were determined to be 35 and 58 mg · kg–1 · d–1, respectively. The results from the current study are the first for lysine requirements in children with the use of the IAAO method. The mean and population-safe requirements for lysine in children are very similar to the adult lysine requirements of 36.6 and 52.5 mg · kg–1 · d–1 determined recently by Kriengsinyos et al (9) and of 36.9 and 58.2 mg · kg–1 · d–1 determined earlier by Zello et al (5) using the IAAO model. The only other study examining lysine requirements in children was by Nakagawa et al (3), who used the nitrogen balance method. They estimated a mean lysine requirement of 60 mg · kg–1 · d–1 in 10-12-y-old boys, which is 41% higher than the current estimate of 35 mg · kg–1 · d–1. The nitrogen balance method has many limitations, including an overestimation of the intake and an underestimation of excretion, which leads to a falsely positive nitrogen balance (2). Indeed, even when lysine intakes were adequate, Nakagawa et al (3) reported nitrogen retention values {approx}5-fold higher than predicted for children of that same age. Also, the nitrogen balance method requires longer periods ({approx}8 d) of adaptation to the test diet (25). For practical reasons, the children in the study by Nakagawa et al (3) were adapted for 3 d to each test intake before the balances were estimated. Thus, during the recent 2002/2005 DRI process (2), the study by Nakagawa et al (3) was not taken into account in determining the recommend mean lysine requirement, and a factorial approach was used.

Lysine requirements in adults have been extensively examined by using the IAAO model (5, 9, 26). In men who were adapted for 2 d to the test lysine intake, the mean lysine requirement was determined to be 36.9 mg · kg–1 · d–1 (5). A similar mean lysine requirement of 36.6 mg · kg–1 · d–1 was determined when the indicator amino acid, L-[1-13C]phenylalanine, was administered intravenously or orally to men (9). In women, the lysine requirements were determined in both phases of the menstrual cycle: 35.0 and 37.7 mg · kg–1 · d–1 for the follicular and luteal phases, respectively (26). The requirement for lysine in children (35 mg · kg–1 · d–1) is very similar to the requirements determined in the abovementioned studies.

Previously, we examined the requirements for total BCAAs and TSAAs in healthy school-age children using the same IAAO protocol (16, 17). The mean total BCAA requirement in children was determined to be 147 mg · kg–1 · d–1 (16), which is similar to the requirement in adults of 144 mg · kg–1 · d–1 (11). The mean TSAA requirement in children was determined to be 12.9 mg · kg–1 · d–1 (17) compared with 12.6 mg · kg–1 · d–1 (10) in adults. In the previous 2 studies in children, we concluded that the similar estimated requirements for children and adults predominantly reflected the maintenance requirements and do not account for all the growth needs for children of this age group. Furthermore, the maintenance nitrogen requirement in children has been reported to be 110 mg · kg–1 · d–1, which does not vary for children aged 9 mo to 14 y and is similar to the nitrogen requirements in adults of 105 mg · kg–1 · d–1 (2). Therefore, to ensure age-appropriate growth in healthy school-age children we proposed that the amino acid requirement for growth should be added to the EAR.

The individual amino acid requirements for growth were recently calculated from the rates of protein deposition for children of different age groups (23, 27), the amino acid composition of whole-body protein (28), and the incremental efficiency of protein utilization (2). The amount of lysine required for growth in 9-13-y-old children was calculated as the product of the rate of protein deposition (49 mg · kg–1 · d–1) and lysine composition of the whole body (0.073%) divided by 0.58, the efficiency of dietary protein utilization, to yield a value of 6.1 mg · kg–1 · d–1. Therefore, the new lysine requirement in children (35 + 6 mg · kg–1 · d–1) is proposed to be 41 mg · kg–1 · d–1. The newly proposed mean and population-safe lysine requirements of 41 and 58 mg · kg–1 · d–1, respectively, are significantly higher than the current DRIs of 37 and 46 mg · kg–1 · d–1, respectively.

In the present study, phenylalanine oxidation (Table 2Go) decreased with increases in lysine intakes, although the differences were significant by Tukey's test only when lysine intakes of 5 mg · kg–1 · d–1 and 35, 65, and 80 mg · kg–1 · d–1 were compared. Conversely, the rate of 13CO2 released from the oxidation of L-[1-13C]phenylalanine (F13CO2) decreased linearly with graded intakes of lysine (Figure 3Go). Breakpoint analysis was performed on F13CO2 data, which has been shown to be a more sensitive endpoint measurement for tracer oxidation in adults (29) and in children (16-18). The ease of collecting breath to measure F13CO2 allowed all children to participate in all test intakes. The repeated-measures model significantly decreased the potential for large intrasubject variation, which has been shown to affect amino acid oxidation (4). The relative noninvasiveness involved in the IAAO method is an advantage and has now been successfully applied in children with inborn errors of metabolism (14, 15) and healthy children (16-18) to determine amino acid requirements. The model can now be applied in other vulnerable populations, such as malnourished children, pregnant and lactating women, and the elderly.

In summary, the EAR for lysine in children was determined to be 35 mg · kg–1 · d–1. This value is similar to the lysine requirement for adults, which suggests that the findings from the present study reflect predominantly the maintenance lysine requirements in children. Therefore, to ensure the age-appropriate growth of school-age children, we propose the addition of a lysine requirement for growth and the new mean and population-safe lysine requirements to be 41 and 58 mg · kg–1 · d–1, respectively. The results of the current study are particularly important because children whose diets consist predominantly of plant proteins and are relatively low in total protein would have inadequate intakes of lysine. The newly proposed higher lysine requirements would enable the appropriate planning of diets and assessment of lysine intakes in children.


   ACKNOWLEDGMENTS
 
We thank all the children who participated in the study and Linda Chow in the Department of Nutrition and Food Services (The Hospital for Sick Children) for preparing the protein-free cookies. We also thank Karen Chapman for coordinating the activities in the Clinical Investigation Unit at The Hospital for Sick Children and Mahroukh Rafii for technical assistance in the laboratory.

The authors' responsibilities were as follows—RE and MAH: study design, data collection, sample and data analysis, and manuscript writing; ROB and PBP: study design, data analysis, and manuscript writing. The authors had no conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication January 19, 2007. Accepted for publication March 21, 2007.




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R. Elango, R. O. Ball, and P. B. Pencharz
Indicator Amino Acid Oxidation: Concept and Application
J. Nutr., February 1, 2008; 138(2): 243 - 246.
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